Bio-resources Technology Division, Forestry and Forest Products Research Institute, Kukizaki, 305, Ibaraki, Japan.
Theor Appl Genet. 1996 Jan;92(1):40-5. doi: 10.1007/BF00222949.
We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5' and 3' ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.
我们使用与 5' 和 3' 端各种长度、序列和非重复基序的简单重复序列退火的引物，研究了花旗松（Pseudotsuga menziesii （Mirb.） Franco）和柳杉（Cryptomeria japonica D. Don）大配子体的 inter-simple sequence repeat（ISSR）多态性和遗传。产物用溴化乙锭染色在琼脂糖凝胶上可视化。在花旗松和柳杉中，测试的 96 个引物中有超过 60%给出了可解释的带型模式，并且有用的引物在物种间完全一致。二核苷酸重复引物是测试的大多数引物，并且给出了所有有用的带型模式。使用 24 个最佳引物进行分离研究，共产生 77 个位点分布在两个花旗松家族和一个柳杉家族中。大约 90%的 24 个引物在至少一个三个家族中显示出多态性。每个引物的可变位点平均数为 1.6。基于（AG）n 重复的引物产生了最多的多态性位点；16 个引物-家族组合产生了 24 个分离的位点。然而，基于（GT）n 重复的引物研究的每个引物具有最多的位点（平均值为 2.0）。所有标记均显示明显的显性（带存在与不存在），除三个分离比（4%）外，所有标记均符合孟德尔预期：由于它们比 RAPD 使用更长的引物，具有高度的多态性，很好地符合孟德尔预期，并且不需要使用丙烯酰胺凝胶进行分析，ISSR 可能是用于基于 PCR 的基因组图谱和针叶树种群研究的有用标记。
